Binding epitopes for g250 antibody

ABSTRACT

The invention relates to specific amino acid sequences which have been determined to be target epitope for antibodies, in particular, for a G250 antibody.

The invention relates to specific amino acid sequences which have beendetermined to be target epitopes for antibodies, in particular, for aG250 antibody.

The G250 antigen is closely associated with numerous carcinomas such asrenal cell carcinoma. The G250 antigen was first described as a kidneycancer-associated antigen (WO 88/08854). Later, it was found to beidentical with the tumor-associated antigen MN, a cell surface antigenwith carbonic anhydrase activity, also referred to as CA-IX.

Normal CA-IX expression is found in gastric, intestinal and biliarymucosa, where its physiological role resides in pH regulation. Besidesits normal expression pattern, CA-IX expression is found in cervicalcarcinomas, esophageal carcinomas, colorectal carcinomas, lungcarcinomas, biliary and clear cell renal cell carcinomas (RCC).

Antibodies against CA-IX, therefore, can be employed for cancer therapy.Anti-G250 antibodies are described, for example, in EP 637 336. Further,WO 02/062972 describes a hybridoma cell line DSM ACC 2526 which producesthe monoclonal antibody G250. The monoclonal antibody G250 recognizes anantigen preferably expressed on membranes of renal cell carcinoma cells(RCC), but not expressed in normal proximal tubular epithelium. The G250antibody binds to the antigen G250, which is also called MN antigen(cf., for example, WO 93/18152) or CA-IX (carbonic anhydrase IX).

The G250 antibody binding site on the CA-IX antigen, however, has notbeen known so far. This made production, reproducibility and recovery ofG250 antibodies difficult, since no specific epitope sequence binding toCA-IX was provided.

Therefore, it was an objective of the present invention to identify thetarget epitope of the G250 antibody.

According to the invention this problem is solved by providing peptidescomprising the amino acid sequence LSTAFARV or the amino acid sequenceALGPGREYRAL or both sequences LSTAFARV and ALGPGREYRAL.

The inventors have found that these peptides constitute the targetepitope of the G250 antibody, and the G250 antibody, accordingly, bindsto said peptides.

The term “G250 antibody”, as used herein, refers to antibodies directedagainst CA-IX antigen and, in particular, to the monoclonal antibodyG250 produced by the hybridoma cell line DSM ACC 2526. Since earlieranalyses suggested that the CA-IX binding site is a conformationalepitope, the peptides of the invention are preferably presented so as tohave the conformational structure as in the protein carbonic anhydraseIX. To this end, they are preferably present in sterically constrainedform. For example, the peptides can be coupled to a carrier to constraina particular conformation.

“Carriers”, as used herein, means both carrier molecules, to which thepeptides can be coupled, as well as articles or surfaces, onto which thepeptides can be applied.

Suitable carrier molecules, for example, are 1,3-di(bromomethyl)phenyl,1,3,5-tri(bromomethyl)-2,4,6-trimethylbenzyl or1,2,4,5-tetra(bromomethyl) phenyl. By means of such carrier molecules asterically constrained geometry of the peptides can be obtained.

It is further preferred that the peptide of the invention is asynthetically produced peptide. Also preferably, it is a non-naturallyoccurring peptide. The sequences of the invention represent the targetepitope of CA-IX, so the peptides preferably represent antigens havingan antigenic effect. Preferably, the peptides of the invention have alength of at least 8, more preferably at least 10, even more preferablyat least 15 and most preferably at least 20 amino acids and up to 300amino acids, more preferably up to 200 amino acids, in particular, up to100 amino acids, even more preferably up to 50 amino acids and mostpreferably up to 30 amino acids. The peptide of the invention is not thecomplete carbonic anhydrase IX polypeptide.

Especially preferably, the peptides comprise any of the followingsequences:

CNQTVCLSTAFARVC, CVPGLDISSCLSTAFARVC,CSPAAAGRFQSPCLSTAFARVC, CLSACLSTAFARVC, CLGPGREYRALC, CGSLTTPPAAQVCLSTAFARVC, CIRPQLAACLSTAFARVC, CHWRYGGDPPWCLSTAFARVC, CLSTAFARVCLSTAFARVC, CALLPSDFSRCLSTAFARVC, CVHLSTAFARVC, ALGPGREYRALQLHL, CLHTLCLSTAFARVC, CALGRPGGCLSTAFARVC,CLGPGREYCLSTAFARVC.

The invention also encompasses peptides comprising the amino acidsequence LSTAFARV and/or the amino acid sequence ALGPGREYRAL, whereby inthe indicated sequences one, two or three amino acids are substituted byother amino acids. Sequences like that can be used as antigens of theG250 antibody as well.

Since the peptides of the invention are antigens of the G250 antibody,they can be used to form a vaccine. It is possible thereby tospecifically administer the antigenic epitope region, preferably inhighly pure form, in order to produce the desired antibody reactionthereby, while at the same time avoiding other reactions, especiallyother immune reactions, since the complete polypeptide CA-IX need not beadministered.

Further, it had been found in the past that purification of the nativeCA-IX antigen is difficult. This was unfavorable, above all, in respectof the provision of assays.

Therefore, another object of the present invention is an assay forpurification of antibodies or binding molecules, comprising (i)providing a peptide of the invention, (ii) contacting a compositioncomprising antibodies or binding molecules with said peptide, (iii)removing non-binding components, and (iv) recovering the antibodies orbinding molecules.

According to the invention an assay is provided, wherein not the nativecarbonic anhydrase IX peptide is applied but an inventive peptide. Insuch an assay, the peptide is preferably coupled to a carrier, forexample, a carrier of plastics, glass or metal. Since the antigenicepitope is presented in the case of the peptides of the invention,antibodies or binding molecules directed against these epitopes bindwith the peptides. Subsequently, non-binding components can be removedand the purified antibodies or binding molecules can then be obtained.The assay for purification according to the invention is especiallysuitable for purification of G250 antibodies or binding molecules.

The invention further comprises an assay for the detection of antibodiesor binding molecules, wherein antibodies or binding molecules directedagainst the inventive peptide which are present in a sample aredetected. To this end, too, a peptide comprising the target epitopes ofthe invention is provided, and it is not necessary to use native CA-IXantigen. A composition possibly containing antibodies or bindingmolecules directed against CA-IX is then contacted with the peptide. Thepeptide is preferably coupled to a carrier in this assay, too. Bindingsto the peptide then can be detected by conventional methods, e.g. bydirect or indirect labeling. In case antibodies or binding molecules arepresent, a positive signal is obtained. Preferably, G250 antibodies orbinding molecules are detected. An assay of that type is suitable, onthe one hand, for the analysis of samples, on the other hand, it mayalso be used for quality control of G250 antibodies.

The advantage that the inventive peptides can be produced in highly pureform in a simple manner and only the antigenic epitopes can be presentedalso can be utilized for a screening assay for identification ofantibodies or binding molecules for CA-IX, comprising (i) providing apeptide of the invention, (ii) contacting candidate antibodies orbinding molecules with said peptide, and (iii) identifying candidatecompounds binding to said peptide as G250 antibodies or bindingmolecules.

Since the invention provides the binding site for G250 antibodies, it ispossible to identify other antibodies binding to G250 (and MN or CA-IX,respectively). To this end, candidate antibodies or binding moleculesare contacted with the peptides of the invention, and candidatecompounds, in the case of which binding is found, can be identified asantibodies or binding molecules for CA-IX. In this way, new G250antibodies can be provided. The invention, therefore, also relates tobinding molecules for the CA-IX antigen which bind to an isolatedsynthetic peptide comprising the amino acid sequence LSTAFARV and/orALGPGREYRAL. The binding molecules thereby are preferably antibodies. Inparticular, the binding molecules are not the G250 antibody produced bythe hybridoma cell line DSM ACC 2526.

New G250 antibodies can be obtained, for example, by a method forproducing or providing an antibody or a binding molecule for CA-IX,comprising (i) providing a peptide of the invention, (ii) generatingantibodies or binding molecules which are able to bind to said peptideby testing for the ability of the antibodies or binding molecules tobind to said peptide.

It has been found that several tumors are CA-IX antigen-expressingtumors, e.g. renal clear cell carcinoma, cervical carcinoma, biliarycarcinoma, esophagus carcinoma, colorectal carcinoma and lung carcinoma.The now identified epitope sequences LSTAFARV and ALGPGREYRAL,therefore, also can be used as markers for the recognition or detectionof carcinoma cells, in particular, for the detection of renal clear cellcarcinoma cells, cervical carcinoma cells, biliary carcinoma cells,esophagus carcinoma cells, colorectal carcinoma cells or lung carcinomacells. Detection of said epitope sequences on a cell characterizes therespective cells as carcinoma cells. Thereby, cells having these markerscan be recognized as cancer cells. On the other hand, it is possible,for example, to detect cancer cells in vivo in tissue by using specificbinding molecules for said markers. Especially preferred is thedetection or recognition of renal cell carcinoma cells.

The identification of the antigenic epitopes of the CA-IX antigen,however, does not only enable diagnostic applications but alsotherapeutic applications. Therefore, the invention also relates to theuse of the amino acid sequence LSTAFARV and/or of the amino acidsequence ALGPGREYRAL as target. The finding that the sequences given inthis invention are specific epitope sequences allows their use astargets, especially as targets in therapy, in particular, for thetreatment of cancer. For example, it is possible to specifically andselectively attack cancer cells by means of antibodies directed againstthe sequences. It is also possible to direct active agentssite-specifically to tumor cells by means of combined agents, e.g. acombination of an antibody specific for these sequences and a furtheranti-cancer agent. For this purpose, the epitope sequence is used as abinding site, either for the therapeutic agent itself or for a targetingaid. Especially preferably, the therapeutic agent itself is an antibodyor binding molecule which binds with the target epitopes and, thus,allows to directly affect cancer cells.

The invention, therefore, also relates to the use of an antibody or abinding molecule for therapy, wherein the antibody or binding moleculeis able to bind to the amino acid sequence LSTAFARV and/or to the aminoacid sequence ALGPGREYRAL.

Knowledge of the inventive target sequences allows to use antibodies orbinding molecules directed against these sequences directly for therapy.

The invention is further illustrated by the appended Figures as well asthe following Examples.

FIG. 1 shows the amino acid sequence of carbonic anhydrase IX.

FIG. 2 shows carrier molecules for forming spatially defined peptides tomimic complex protein structures.

FIG. 3 shows the localization of the epitope sequences LSTAFARV andALGPGREYRAL according to the invention. Both regions form a clearlyexposed discontinuous epitope on CA-IX.

EXAMPLES Example 1 Identification of Target Peptides

Short peptide sections of CA-IX which bind to the G250 antibody weredetermined by means of conformational epitope mapping of carbonicanhydrase using the scaffolds shown in FIG. 2. The following 15sequences were identified as peptide sections having the highest bindingcapacity:

CNQTVCLSTAFARVC, CVPGLDISSCLSTAFARVC,CSPAAAGRFQSPCLSTAFARVC, CLSACLSTAFARVC, CLGPGREYRALC, CGSLTTPPAAQVCLSTAFARVC, CIRPQLAACLSTAFARVC, CHWRYGGDPPWCLSTAFARVC, CLSTAFARVCLSTAFARVC, CALLPSDFSRCLSTAFARVC, CVHLSTAFARVC, ALGPGREYRALQLHL, CLHTLCLSTAFARVC, CALGRPGGCLSTAFARVC,CLGPGREYCLSTAFARVC.

The sequence LSTAFARV is dominantly recognized, followed by the sequenceALGPGREYRAL.

Example 2 Localization of Identified Epitope on CA-IX

The localization of the identified target sequence sections was shown ona three-dimensional model of CA-IX (cf. FIG. 3).

Example 3 Characterization of G250 Antibody Binding Site

The CA-IX protein is composed of a large extracellular domain (ECD), asingle-path transmembrane region (TM) and a short intracellular tail(IC). ECD consists of an N-terminal proteoglycan-like region (PG) and awell-conserved, catalytically active carbonic anhydrase (CA) domain. Forbetter understanding of G250 antibody properties, the location of itsbinding site was analyzed.

For this, MDCK canine kidney cells permanently transfected with thefull-length CA-IX cDNA in pSG5C plasmid (MDCK-MN) or with plasmidSderived therefrom encoding a dCA variant (MDCK-dCA), a dPG variant(MDCK-dPG) or hAS (human alternatively spliced protein truncated in theC-terminal part of the CA domain, MDCK-hAS). Mock transfected cells(MDCK-neo) were used as a negative control. A dCA variant means a CAdomain deleted variant.

The binding of G250 antibody to the various cells was tested byincubating microplate wells which were coated with the respective cellseach with G250 antibody. Peroxidase-labeled pig anti-mouse IgG was usedas detector.

The obtained results showed that G250 antibody binds to a conformationalepitope localized in the CA domain of CA-IX.

1-22. (canceled)
 23. A peptide comprising the amino acid sequenceLSTAFARV and the amino acid sequence ALGPGREYRAL, wherein the peptide isnot carbonic anhydrase IX.
 24. The peptide of claim 23, wherein thepeptide is a synthetic peptide.
 25. The peptide of claim 23, wherein thepeptide is sterically constrained.
 26. The peptide of claim 23, whereinthe peptide is an antigen.
 27. The peptide of claim 23, wherein thepeptide is coupled to a carrier.
 28. A vaccine comprising a peptide ofclaim
 23. 29. An assay for purification of antibodies or bindingmolecules, comprising: providing a peptide according to claim 23,contacting a composition comprising antibodies or binding molecules withsaid peptide, removing non-binding components, and recovering theantibodies or binding molecules.
 30. The assay of claim 29 forpurification of G250 antibodies or binding molecules.
 31. An assay forthe detection of antibodies or binding molecules, comprising providing apeptide according to claim 23, contacting a composition suspected tocontain antibodies or binding molecules with said peptide, and detectingcomponents of the composition binding to said peptide.
 32. The assay ofclaim 31 for the detection of G250 antibodies or binding molecules. 33.A screening assay for identification of antibodies or binding moleculesfor CA-IX, comprising providing a peptide according to claim 23,contacting candidate antibodies or binding molecules with said peptide,and identifying candidate compounds binding to said peptide as G250antibodies or binding molecules.
 34. A binding molecule for the CA-IXantigen, wherein said binding molecule binds to an isolated syntheticpeptide comprising the amino acid sequence LSTAFARV and ALGPGREYRAL,wherein the binding molecules are not the G250 antibody produced by thehybridoma cell line DSM ACC
 2526. 35. The binding molecule of claim 34,wherein the binding molecule is an antibody.
 36. Use of the amino acidsequence LSTAFARV and of the amino acid sequence ALGPGREYRAL as a markerfor the recognition or detection of carcinoma cells.
 37. Use of theamino acid sequence of claim 36 for the detection or recognition ofrenal cell carcinoma cells.
 38. Use of the amino acid sequence LSTAFARVand of the amino acid sequence. ALGPGREYRAL as a target.
 39. Use of theamino acid sequence of claim 38 as a target for therapy.
 40. Use of theamino acid sequence of claim 38 as target for treatment of cancer. 41.Use of the amino acid sequence of claim 38, wherein the amino acidsequence constitutes a binding site for a therapeutical agent.
 42. Useaccording to claim 41, wherein the therapeutical agent is an antibody orbinding molecule capable of binding to a peptide as defined in claim 23.43. Use of an antibody or a binding molecule for therapy, wherein theantibody or binding molecule is bindable to the amino acid sequenceLSTAFARV and to the amino acid sequence ALGPGREYRAL.
 44. A method forproducing or providing an antibody or a binding molecule for CA-IX,comprising providing a peptide according to claim 23, generatingantibodies or binding molecules, wherein said antibodies or bindingmolecules are bindable to said peptide; said generating furthercomprising testing the ability of said antibodies or binding moleculesto bind to said peptide.